The ectoplasmic specialization (ES) of Sertoli cells is believed to play a critical role in spermatogenesis. This intercellular junction is thought to anchor and position spermatids within the seminiferous epithelium throughout much of spermiogenesis and may also contribute to the "blood-testis" barrier between Sertoli cells. These activities are believed to depend on the ES junctional plaque which is, characterized by a narrow layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum. The long-term objectives of this project are to determine how the ES is organized at the molecular level and to define the roles of the ES and its protein components in spermatogenesis and fertility. To this end, we have recently identified and characterized "espin," a novel approximately 11O-kDa protein that is a major chemical constituent of the ES (approximately 5 million copies/ES) localized to the parallel actin bundles of its junctional plaque. The localization, stoichiometry and tissue distribution of espin, together with the actin-binding/bundling properties displayed by its C-terminal half in vitro and in transiently transfected cells, suggest that espin is a major ES-specific actin- bundling protein responsible for the formation of the parallel actin bundles that comprise this key layer of the ES junctional plaque. Since espin is a novel protein that is likely to play a central role in the organization and function of the ES, it will be the focus of the proposed experiments. Specifically, we propose: (1) to test the hypothesis that espin is an actin-bundling protein and to determine how the N-terminal half of espin affects the properties of the protein by examining the actin-binding/bundling properties of full-length espin in vitro and in vivo in transiently transfected or microinjected cell models; (2) to provide new information about the molecular organization of the ES and to test the hypothesis that espin is involved in the formation of the actin bundles of the junctional plaque by localizing espin in relation to other ES proteins and ultrastructural landmarks during the assembly and disassembly of Ess that accompanies spermatogenesis; (3) to test the hypothesis that espin and the ES play critical roles in spermatogenesis by examining the consequences of eliminating espin expression by gene targeting.